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Basic study on flurbiprofen protein binding inhibition by an albuminsite II binding inhibitor, 6-methoxy-2-naphthyl acetic acida)Jin Tokunaga Norito Takamura Kenji Ogata
Teruyoshi Kondo*Hiroki Yoshida Nao Setoguchi

Toyotaka Nishio** Keiichi Kawai**

Abstract
The protein binding rate of a nonsteroidal antiinflammatory drug (NSAID), flurbiprofen (FP), is about
99%, indicating its low tissue transfer. 6-Methoxy-2-naphthyl acetic acid (6MNA) is an active metaboliteof an NSAID, nabumetone (Relifen ®). These drugs strongly bind to human serum albumin (HSA) siteII, suggesting that protein binding inhibition by 6MNA may promote tissue transfer of FP. We
investigated cell membrane permeability of FP in the presence of 6MNA using swine kidney cell line
LLC-PK1, in which the upper and lower sides of the cell membrane plate (Transwell ®) were regarded
as the tissue and vascular sides, respectively. After adding 6MNA to HSA solution containing FP on
the lower side, the FP levels in the upper and lower solutions and cells were measured. FP transfer tothe upper side was significantly promoted by the addition of 6MNA with regard to the total and freeFP levels. No change in the FP level was noted in cells or wells. It was suggested that concomitantnabumetone may promote transfer of site II binding drugs, such as FP, to the target tissue, increasing
the effect even at a low dose.

Key words： human serum albumin, protein binding, site II, flurbiprofen, 6-methoxy-2-naphthyl
acetic acid2009.11.17

accept(AGP) molecules and laboratory test values affecting theINTRODUCTION

binding, and established a simple diagnostic method ofThe serum protein binding rate of a nonsteroidal

pharmacological distribution to determine the timing ofantiinflammatory drug (NSAID), flurbiprofen (FP), isdrug administration and increase the pharmacological1)very high (about 99%), indicating its low tissue transfer .

effect3-6). We also reported that protein binding inhibition

6-Methoxy-2-naphtyl acetic acid (6MNA) is an active

by a free fatty acid (FFA), oleic acid (Ole), elevated themetabolite of an NSAID, nabumetone (Relifen ). Its

blood FP level2). However, we have not investigated cell®

biological half-life is long, and the therapeutic

membrane permeability involved in tissue transfer ofconcentration is high. These drugs strongly bind to site

the drug, and only reported the protein binding-

II of human serum albumin (HSA) 2). Thus, we consideredinhibitory phenomenon and pharmacokinetic changes inthat protein binding inhibition by 6MNA may promote

the body. We previously performed a basic study on celltissue transfer of FP and increase its effect at a low

membrane permeability of probe drugs in the presence

dose. We previously measured the drug binding abilityof protein binding inhibitors using cells cultured onto binding sites on HSA and α 1-acidic glycoprotein

plates for cell permeation, and established this method7).

九州保健福祉大学 薬学部 〒882-8508 宮崎県延岡市吉野町1714-1
School of Pharmaceutical Sciences, Kyushu University of Health and Welfare, 1714-1 Yoshino-machi, Nobeoka-shi, Miyazaki, 882-8508 Japan*九州保健福祉大学 保健科学部*
School of Health Science, Kyushu University of Health and Welfare**
金沢大学大学院 医学研究科**
Kanazawa University Graduate School of Medical Science

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Kyushu University of Health and Welfare154九州保健福祉大学研究紀要

Thus, using swine kidney cell line LLC-PK1, we

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3. Setting of HSA concentration

investigated cell membrane permeability of FP in thepresence of 6MNA.

The culture medium was changed to EBSS on
experiment day (1.5 and 2.6 mL on the upper andlower sides, respectively). FP (final concentration: l40

MATERIALS AND METHODSμ M) was added to HSA solution at various
concentrations (final concentration: l0, 40, 100, 200,7)

1. Preparation of LLC-PK1 cell membrane plates

400, and 600 μM), and the total and free FP levels on

Commercial swine kidney-derived LLC-PK1 cells

the upper and lower sides were measured every hour

were seeded in a culture flask (25 cm2) containing cellfor 4 hours (free FP was measured only at 4 hours).

growth medium (Earles Medium 199, GIBCO)

To measure free FP, the upper and lower solutions

supplemented with antibiotics (penicillin-streptomycin,

were centrifuged at 3,000 rpm for 10 minutes andGIBCO) and fetal bovine serum (FBS, GIBCO). The

ultrafiltrated (TOSO) to prepare free FP solutions. Thecells were cultured in a CO2 incubator (5% CO2 -95%

protein binding rate of FP was calculated from theair, 37℃). The medium was changed following day,total and free FP levels.

and the cells were further cultured for 5-6 days withchanging medium and culture flask (75 or 150 cm2)

4. Cell membrane permeation experiment of FP

every 2 days. After culture, the cells were detached

The culture medium was changed to EBSS on

and suspended with Trypsin-EDTA solution (0.05%

experiment day (1.5 and 2.6 mL on the upper andtrypsin, 0.02% EDTA, GIBCO), seeded in 6-well plates

lower sides, respectively). 6MNA solution (finalfor cell permeation (membrane pore size: 3.0μm,concentration: 50 and 100μM) was added to the HSA

Transwell , CORNING) at 2 x 10 cells per well, andand FP solutions at the final concentrations

cultured for 3 days. The upper and lower sides of theestablished on setting the HSA concentration: 200 μM

cell membrane plate were regarded as the tissue andand 40μM, respectively, and the total and free FP

vascular sides, respectively. Earles M199 Solution

levels in the upper and lower solutions were(GIBCO) was used for culture and Earle s Balanced

measured after 2 and 4 hours (free FP was measured

Salt Solution (EBSS, GIBCO) for setting of the HSA

only at 4 hours). In addition, the intracellular FP level

concentration and cell membrane permeation

was measured after the experiment. To measure®

6

experiment of FP. LLC-PK1 cells were passaged 15-18

intracellular FP, the membrane was detached from thetimes before the experiment. The cell membrane

plate and washed twice with EBSS in a microtube,

permeation experiment of FP was triplicate orEBSS was removed by sucking, and cells werequadruplicate (n=3-4).

dissolved with an equal volume of trypsin (10 x) and2N NaOH, followed by centrifugation at 3,000 rpm for

2. FP measurement method2)

10 minutes. The supernatant was then neutralized

FP was measured using high-performance liquid
chromatography (HPLC). All samples excluding freewith an equal volume of 1N HCl and subjected to FP
measurement.

FP solution were deproteinized with methanol before
injection into HPLC. The HPLC measurement
conditions for FP analysis were as follows:

5. Statistical analysis
All values were presented as the means±standard®

HPLC, SHIMADZU CLASS-VP; column, LiChrospherdeviation. For analysis of significance of differences in

RP-18 Select B (5 μm), 250 mm L. x 4.0 mm I.D.;

the FP levels in the upper and lower solutions,mobile phase, 0.1 M citric acid - 0.1 M sodium acetate

analysis of covariance was employed, and Fisher s(pH 4.5): acetonitrile=1:1; flow rate, 1.0 mL/min;

PLSD was used for multiple comparison. For analysis

column temperature, 40℃; injection volume, 30 μL;of significance of differences in the free andexcitation, 248 nm; and emission, 320 nm.

intracellular FP levels, non-repeated measures
ANOVA was employed, and Dunnett s test was used

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for multiple comparison. p<0.05 was regarded assignificant.

RESULTS
1．Setting of HSA concentration
To evaluate cell membrane permeability of FP in
the presence of 6MNA, the HSA concentration wasestablished (0, 40, 100, 200, 400, and 600 μM). The

Table 1 The protein binding rate (%) of FP in the lowersolution with changes in the HSA concentration

FP levels in the upper solution between 0 and 4

The values are presented as the means±

hours are shown in Fig. 1. The HSA addition

standard error.

markedly reduced the level. The FP levels in thelower solution at 4 hours are shown in Fig. 2. The
HSA addition markedly increased the level. The

2. Cell membrane permeation experiment of FP

protein binding rates of FP at 4 hours in the lower

The total FP levels in the upper solution at 0-4solution are shown in Table 1. The protein binding

hours are shown in Fig. 3. The total FP level in therate rose as the HSA concentration increased.

upper solution was significantly increased at 4hours in the presence of 50μM 6MNA (p<0.05), but
the total level in the presence of 100 μM 6MNA
was lower than that in presence of 50μM 6MNA.
The total FP levels in the lower solution at 0-4
hours are shown in Fig. 4. The total level in thelower solution was not changed at 2 or 4 hours.
The free FP levels at 4 hours are shown in Fig. 5.
The free FP level in the upper solution wassignificantly elevated by the addition of 100μM

Fig. 1 The FP level (μg/mL) in the upper solution withchanges in the HSA concentration

6MNA at 4 hours (p<0.01). No significant change
was noted in the free FP level in the lowersolution or in the intracellular level at 4 hours (Fig.
6).

Fig. 3 The FP level (μg/mL) in the upper solution in
Fig. 2 The FP level (μg/mL) in the lower solution withchanges in the HSA concentration

the presence of the inhibitor
The values are presented as the means±standard error (n=4).
*; p<0.05

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DISCUSSION
Cell membrane permeability of FP in the presenceof 6MNA was investigated using swine kidney cellline LLC-PK1. The addition of 6MNA significantly
elevated the total and free FP levels.
Since the blood HSA level is about 600μM, the cellFig. 4 The FP level (μg/mL) in the lower solution inthe presence of the inhibitor
The values are presented as the means±standard error (n=4)

membrane permeability experiment of FP should be
investigated at an HSA concentration of 600μM.
However, HSA is a useful human-derived reagent.
Thus, we investigated the HSA concentration atwhich the protein binding rate of FP can be
maintained at 99%, and performed the experimentat the minimum HSA concentration. A high protein
binding rate of FP was confirmed, as reported byTohno et al.1), and the protein binding rate of FP
was 99.9% at HSA concentrations of 200, 400, and600μM. Based on these, the HSA concentration
was set at 200 μM in this experiment.
Cell membrane permeability of FP in thepresence of 6MNA was investigated using LLCPK1 cells. The 6MNA addition significantlyFig. 5 The free FP levels (μg/mL) in the upper andelevated the total and free FP levels, showing thatlower solutions in the presence of the inhibitor

cell membrane permeability of FP was increased.

The values are presented as the means±standard

6MNA may have prevented FP from protein

error (n=4). **; p<0.01.

binding by binding to HSA site II, which elevated
the free FP level in the lower solution andpromoted their transfer to the upper solution.
The total FP level in the upper solution at 4hours in the presence of 100 μM 6MNA was lower
than that in the presence of 50 μM 6MNA as
shown in Fig. 3. We considered its reason as
follows: The total FP level in the upper solution in
the presence of 100 μM 6MNA tended to be higher
than that in the presence of 50 μM 6MNA at 2hours as shown in Fig. 3, and the free FP level inFig. 6 The intracellular FP level (μg) in the presence ofthe inhibitor
The values are presented as the means±standard error (n=4).

the upper solution was significantly higher thanthat in the presence of 50μM 6MNA at 4 hours asshown in Fig. 5. The total FP level in the uppersolution at 4 hours should have been higher in thepresence of 100 μM 6MNA, but free FP may havetransferred to the lower solution due to its
increased level, reducing the total FP level in theupper solution. However, no significant changes

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were noted in the total or free FP level in theconstruction of the most unique bedside

lower solution at 4 hours as shown in Figs. 4 andtraining, Yakuzaigaku 67: 165-171, 2007.

5, respectively.

4) Takamura, N., Tokunaga, J., Chosa, E., et al.:Kawai et al. recently reported that scintigraphy
with a radioactive imaging diagnostic agent,

123

I-N-

isopropyl-p-iodoamphetamine (IMP), could be

Need for pharmaceutical skill in using
displacement of protein binding, Yakugaku
Zasshi 127: 1805-1811, 2007.

8)

performed at a low dose using 6MNA in monkeys .

5) Takamura, N., Tokunaga, J., Arimori, K.:

Both IMP and 6MNA bind to albumin site II. They

Interpretations of laboratory test data on

utilized the increase in the free IMP level by priorserum protein binding, Yakugaku Zasshi 127:binding of 6MNA to albumin site II. Their finding

231-236, 2007.

may have been due to increased cell membrane6) Takamura, N., Maruyama, T., Chosa, E., et al.:permeability demonstrated in our study: inhibition

A potent binding inhibitor for furosemide,of binding to albumin in blood vessels may havealters the pharmacokinetics and diuretic effectpromoted tissue transfer of IMP.

of furosemide: potential for use of bucolome to6MNA increased cell membrane permeability of

restore diuretic response in nephrotic

FP by inhibiting protein binding of FP, suggesting

syndrome, Drug Metab. Dispos. 33: 596-602,

that concomitant nabumetone (prodrug of 6MNA,2005.

Relifen ®) enhances transfer of site II-binding drugs,7) Tokunaga, J., Takamura, N., Ogata, K., et al.:such as FP, to the target tissue, increasing theBasic examination of the cell membrane

effect at a low dose. The series of our findings

permeability of site probe agents using a cellsuggest that adequate use of safe protein binding

membrane plate, J. Kyushu Univ. Healthinhibitors may promote tissue transfer. We areWelfare 10: 193-199, 2009.

planning to develop a dose reduction method for8) Kawai, K., Nishii, R., Shikano, N., et al.: Serumhigh-risk drugs based on these basic studies andprotein binding displacement: theoretical analysis

evidence obtained from various human cells.

using a hypothetical radiopharmaceutical a n dexperimental analysis with 123 I-N-isopropyl-p-a)This study was partially supported by researchiodoamphetamine, Nucl. Med. Biol. 36: 99-106,

grant from the aluminum association of2009.

Kumamoto University School of Pharmacy.

REFERENCES1) Tohno, M., Konishi, Y., Kojima, H., et al.:Metabolism and disposition of LFP83 in man
and other several species after intravenous
administration, Kiso to Rinsyo 22: 4031-4045,1988.
2) Ogata, K., Takamura, N., Tokunaga, J., et al.:Dosage plan of a flurbiprofen injection productusing inhibition of protein binding by lipid
emulsion in rats, J. Pharm. Pharmacol. 60: 1520, 2008.
3) Takamura, N., Tokunaga, J.: The pharmaceutical
distribution diagnostic method which wascreated by clinical sense of a pharmacist: ANII-Electronic Library ServiceKyushu University of Health and Welfare

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九州保健福祉大学研究紀要

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アルブミンサイトⅡ結合阻害薬である6-メトキシ-2-ナフチル酢酸によるフルルビプロフェンの蛋白結合阻害に関する基礎的検討a）
徳永仁

緒方賢次

0村 徳人 吉田 裕樹*近藤 照義**西尾 豊隆 **川井 恵一

瀬戸口奈央

九州保健福祉大学薬学部
九州保健福祉大学保健科学部*

金沢大学大学院医学研究科

**

日本語要旨
非ステロイド性抗炎症薬（NSAID）フルルビプロフェン（FP）の蛋白結合率は約99%であり、組織移行性が低い。6-メトキシ-2-ナフチル酢酸（ 6MNA）は、NSAIDの1つであるナブメトンの活性代謝産物である。これらの薬物は、ともにヒト血清アルブミン（HSA)のサイトⅡに強く結合する。
私たちは、LLC-PK1を用いて6MNA添加時のFPの細胞膜透過性について調べた。この実験では、細胞膜プレートの上側を組織側、下側を血管側と想定した。下側のFPを含むHSA溶液に6MNAを添加
後、上側、下側のFP濃度を測定した。上側へのFPの移行量は6MNAの添加により、Total FPおよびFree FP濃度ともに有意な増加を示した。下側の量は変化が見られなかった。臨床におけるナブメトンの併用はFPのようなサイトⅡ結合性薬物の標的組織への移行量を増加させ、少量においても効果を高める可能性が示唆された。
キーワード：ヒト血清アルブミン, 蛋白結合, サイトⅡ, フルルビプロフェン, 6-メトキシ-2-ナフチル酢酸

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